Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in non-tuberculous mycobacteria isolates from Kenya

Background: Non-tuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing Mycobacteria (RGM), and rifampicin for Slow-Growing Mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes including rrl, rrs, and rpoB in NTM isolates from Kenya. Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene. Results: NTM harboring mutations associated with resistance to at least one of the antibiotics used in the macrolide-based therapy were 23% (28/122). Of these NTM, 10.4% (12/122) had mutations in the rrl gene with 58.3% (7/12) comprising RGM and 41.7% (5/12) being SGM. Mutation at position 2058 (A2058G, A2058C, A2058T) of the rrl gene was seen for 83.3% (10/12) of NTM, while 16.6% (2/12) harbored a A2059G mutation. Of the 54 RGM included for rrs characterization, 11.1% (6/54) exhibited mutations at position 1408(A1408G), while 14.7% (10/68) of the SGM had mutations in the rpoB gene at positions S531W, S531L, S531Y, F506L, E509H with M.gastri having multiple mutations at positions D516V, H526D and, S531F. Conclusion: We demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya.

. In mycobacterial culture using Lowestein-Jensen (LJ) media which is a solid selective media for mycobacteria, NTM varies in their growth characteristics, with Rapid Growing Mycobacteria (RGM) forming visible colonies within seven days of incubation and Slow-Growing Mycobacteria (SGM) taking up to fourty days. SGM include Mycobacterium avium complex (MAC), Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium malmoense, and Mycobacterium xenopi, whereas RGM comprise species from the Mycobacterium abscessus and Mycobacterium fortuitum complexes (Alffenaar et al., 2021). In human infections, identifying NTM is crucial for determining clinically relevant species and the best treatment plan (Chalmers et al., 2019;Mwangi et al., 2022a).
NTM treatment consists of a macrolide-based antibiotic regimen which include clarithromycin or azithromycin, combined with other selected antibiotics which are given for 12 to 18 months until culture negativity is achieved. (Falkinham, 2018). In addition to macrolides, the antibiotic of choice is largely determined by the infecting NTM, its growth rate, and the intricacy of the mycolic acid cell wall (Goswami et al., 2016). Rifampicin and ethambutol are also used in SGM treatment, while aminoglycosides, cefoxitin, imipenem, or tigecycline are used in RGM treatment (Brown-Elliott et al., 2012;Saxena et al., 2021). Due to the slow growth of NTM compared to other bacterial infections, antimicrobial combination therapy is strongly recommended in NTM treatment to avoid the development of drug resistance (Falkinham, 2018;Pharmd et al., 2019). The antibiotic regimen of patients with pre-existing lung diseases including asthma, chronic obstructive pulmonary disease, cystic fibrosis, and bronchiectasis or with immunodeficiencies are the same as for patients without these comorbidities (Koh, 2017).
Anti-mycobacterial drugs attach to their binding sites with a high affinity, preventing the target gene product from functioning normally (Alffenaar et al., 2021). Changes in the structure of the target regions caused by mutations, on the other hand, interfere with the medications' ability to attach to them, resulting in antibiotic resistance. As a result, determining the antibiotic resistance profile of NTM is critical for determining an effective treatment strategy for a specific NTM infection (Goswami et al., 2016;Nasiri et al., 2017).
Acquired resistance to anti-NTM drugs develops due to mutations in the NTM drug target regions, subsequently promoting NTM evolution to mutant strains that are insusceptible to anti-NTM drugs (Huh et al., 2019;Munita & Arias, 2016;Nasiri et al., 2017;Pharmd et al., 2019;Saxena et al., 2021). Prolonged exposure to NTM antibiotics, as seen in the lengthy NTM regimen, sub-optimal administration of anti-NTM drugs, as seen in patients who do not adhere to the regimen or who are lost to follow up, and incorrect prescription for NTM infection due to NTM misdiagnosis all promote mutation in the drug target regions (Gopalaswamy et al., 2020;Munita & Arias, 2016;Zhou et al., 2020).
Macrolides inhibit protein synthesis by binding to the peptide exit tunnel of ribosomes, hence preventing the growing peptide chain from exiting the peptidyl transferase center of the ribosome (Hansen et al., 2002). Mutation of the rrl gene at positions A2058 and A2059 accounts for 80-100% of the macrolide resistance in NTM. Macrolide resistance can also be conferred by the erm (41) gene which encodes a ribosomal methyltransferase that methylates the rrl thus blocking the drug-binding site of the macrolide (Bastian et al., 2011;Huh et al., 2019).

REVISED Amendments from Version 4
Title: Inclusion of a hyphen in Non-tuberculous Abstract: Inclusion of a hyphen in Non-tuberculous Mycobacterium (NTM). Used upper case for the first letters of the acronym SGM and RGM. In the results section, the percentage are outside the brackets while proportions have been indicated in brackets. For mutations, nucleotide change has been indicated for each position. Introduction: Details of Lowenstein Jensen media for growth of mycobacteria has been included. Second paragraph, the duration of treatment of NTM infections has been indicated. indication that Rifampicin is recommended for first-line treatment of MTB alongside other drugs. Laboratory procedures: Sputum sample estimate volume for each participant has been provided. Details on FM microscopy readers and reviewers is now indicated. Results: The table with results indicating that the bulk of the samples were obtained from Lake Victoria, Coastal and Nairobi region has been mentioned. Discussion: rrl significant mutations are critically discussed and their relevance to macrolide drug resistance.
Any further responses from the reviewers can be found at the end of the article Aminoglycosides inhibit protein synthesis by binding the bacterial 30S ribosomal subunit interfering with bacterial protein translation and leading to cell death (Saxena et al., 2021). Drug resistance to aminoglycosides is associated with modification in the rrs gene mainly observed as point mutation at position 1408 (Brown-Elliott et al., 2013). In addition, mutations at positions 1406, 1409, and 1491 have been shown to confer resistance to aminoglycosides in some NTM (Olivier et al., 2017).
Rifampicin is a key drug in treating mycobacterial diseases. It is the recommended first line drug for treatment of M. tuberculosis alongside other drugs (WHO, 2014). It inhibits the synthesis of Ribonucleic acid (RNA) by binding to the ß-subunit of the RNA polymerase that is encoded by rpoB gene. Most rifampicin-resistant mycobacteria have mutations in an 81-bp rifampicin resistance determining region (RRDR) within the rpoB gene. Mutations in this region account for 95% of rifampicin resistance in mycobacteria. The commonly observed mutations within the RRDR of rpoB are often seen at codons 526, and 531 (Li et al., 2016). M. kansasii has also shown mutation conferring resistance to rifampicin at codons 513 and 516, while MAC also shows resistance to rifampicin with mutations outside the RRDR at codon 626 (K626T) (Ramasoota et al., 2006).
We, therefore, investigated drug resistance in NTM by describing the mutation patterns in rrl, rrs, and rpoB genes for macrolides, aminoglycosides and rifampicin respectively, in NTM isolated from symptomatic TB-negative patients from Kenya.

Methods
We carried out a cross-sectional study that included 122 NTM identified by mycobacterial culture and hsp65 targeted sequencing from the sputum of symptomatic TB-negative patients (Mwangi et al., 2022a). The patients had provided approximately 5 ml of sputum to the National Tuberculosis Reference Laboratory (NTRL) in Kenya between January to November 2020.
In this study, the 122 NTM including 54 RGM and 68 SGM underwent targeted sequencing of the rrl gene. The RGM were also sequenced for rrs, and the SGM were sequenced for rpoB genes using ABI 3730XL analyzer (Applied Biosystems, Foster City, California, USA).

Laboratory procedures
Sample processing, mycobacterial culture and growth identification The sputum samples were decontaminated using the N-acetyl-L-cysteine 2% NaOH (NALC-NaOH) procedure, then inoculated into Mycobacteria Growth Indicator Tube (MGIT) and Lowestein-Jenseen (LJ) medias, incubated at 37°C and monitored for growth for up to eight weeks respectively. At the same time, sputum smears were prepared, air dried, heat fixed then fluorochrome stained with auramine O where mycobacteria appeared as bright yellow fluorescent rods when viewed under a light emitting diodes (LED) microscope. Fluorescent microscopy was performed by NTRL laboratory technologist then smears were reviewed by the section head before reporting.
The culture growth in MGIT and LJ underwent the Mtb identification testing using the SD Bioline TB Ag MPT64 assay (capilia) (Standard Diagnostics, Yongin-si, Gyeonggi-do, Republic of Korea) and capilia positive samples were excluded from the study. The capilia negative samples underwent ZN microscopy with presence of AFB indicating a possible NTM.

DNA extraction
Mycobacterial DNA was extracted from 500 μL of re-suspended colonies using using GenoLyse ® (Hain Lifescience, Nehren, Germany) according to the manufacturer's instructions. Briefly, 100 ul of lysis buffer (A-LYS) buffer was added to each cryovial containing the resuspended colonies and incubated for five minutes at 95 o C after which 100 ul neutralization buffer (A-NB) was added and centrifugation done at 5000G for ten minutes. The supernatant was transferred to a newly labelled cryovial awaiting PCR.
Conventional PCR, DNA gel electrophoresis and DNA purification Conventional PCR targeting rrl, rpoB and rrs genes of NTM were conducted using the Horse-Power™ Taq DNA Polymerase MasterMix (Canvax, Córdoba, Spain) in a final reaction volume for each gene of 13 μl comprising 6.25 μl of 2X Horse-Power™ Taq DNA Polymerase MasterMix, 2.5 μl DNA template, 0.25 μl of each of both forward and reverse primers (Table 1) at a final concentration of 10 pmoles, and 3.75 μl of nuclease-free water to make up the reaction volume. The PCR assays were carried out with a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) ( Table 1). Thermal cycling conditions for rrl were as follows: one cycle of 95°C for five minutes, 35 cycles of 95°C for one minute, 55°C for one minute, 72°C for one minute, and a final extension for ten minutes at 72°C. PCR for rrs was conducted as follows 95°C for five minutes, 35 cycles of 95°C for one minute, 60°C for one minute, 72°C for one minute, and a final extension for seven minutes at 72°C. PCR for the rpoB was conducted as follows: 95°C for five minutes, 35 cycles of 95°C for one minute, 58°C for one minute, 72°C for one minute, and a final extension for seven minutes at 72°C. Amplified products were confirmed on a 1% Agarose gel stained with 4.6 μl SYBR safe DNA stain (Invitrogen, Carlsbad, California, USA), and results were visualized with an UltraViolet gel viewer (Terra Universal, S. Raymond Ave, Fullerton, CA, USA). For the corresponding runs, a positive control containing drug-resistant NTM with mutations in the rrl, rpoB and rrs genes was used, as well as a negative control containing drug sensitive NTM without mutations in the drug target genes were included.
The PCR products were enzymatically purified using ExoSAP IT (Applied Biosystems, Foster City, California, USA). Purification conditions were done at 37°C for fifteen minutes followed by a second incubation at 80°C for fifteen minutes and a final cooling step at 4°C for five minutes.

rrl, rpoB and rrs genes sequencing
The purified amplicons were sequenced in the forward and reverse directions by Sanger sequencing using Big Dye™ Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) and the forward and reverse primers. The sequencing reaction for each gene was a 10 μl reaction comprising 1.25 μl of Big Dye Terminator, 3 μl of 5Â Sequencing Buffer, 1 μl of 1 pmol of the sequencing primer, and 1.5 μl of the PCR product. The reaction volume was made up by adding 3.25 μl of nuclease-free water. The reaction proceeded through 96°C for 1 minute then 25 cycles of 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes.
Purification of cycle-sequencing products was done using the BigDye X Terminator™ purification kit following the manufacturer's instructions (Applied Biosystems, Foster City, California, USA) and purified products were loaded onto the ABI 3730 genetic analyzer (Applied Biosystems, Foster City, California, USA) for capillary electrophoresis.

Data analysis
The obtained sequences were first assembled into contigs and the consensus sequences aligned to their wild-type reference sequences for each gene using Geneious version 11.0 (Biomatters Ltd, Auckland, New Zealand). Mutations in the drug resistance genes were identified visually. STATA version 14 (StataCorp, College Station, Texas, USA) was used to test the association of NTM species to mutation patterns using Pearson chi-square at 95% confidence interval.

Results
Our study established that 23% (28/122) of NTM harbored mutations associated with resistance to at least one of the antibiotics in the macrolide-based therapy. One isolate (C47) containing M. abscessus had mutations conferring resistance in both rrl and rrs genes.
The bulk of the isolates with mutations associated with drug resistance originated from the Lake Victoria, Coastal, and Nairobi regions, with 5% (6/122) NTM showing mutations in the rrl, rrs, or rpoB genes (p=0.012)) ( Table 2). The age group was statistically significant (p=0.012), with isolates from participants aged 21 to 35 years old having the highest (n=10, 35.7%) number of NTM with target gene alterations for rrl, rrs, and rpoB genes. Males accounted for 76.5% (21/28) of the NTM isolates with mutations in the drug target genes and indicated a strong statistical significance (p=0.000). The majority of NTM with these mutations were detected in new (n=8; 28.5%) and TB relapse patients (n=8; 28.5%) patients, which had a significant statistical significance (p=0.001) ( Table 2).  Mutations in rrl gene for the NTM were highly significant with a p value of <0.001. Of the NTM with mutations in their drug target genes, 10.4% (12/122) had mutations in the rrl gene (Table 2) with 58.3% (7/12) comprising RGM (Table 3) and 41.7% (5/12) being SGM (Table 3) (Table 3).
A low association (p=0.89) for rpoB mutation in SGM was observed. Mutations within codons between 503-533 of the rpoB were seen for 14.7% (10/68) of SGM. These SGM included M. avium subsp avium, (S531W S531L, S531Y) M. intracellulare (F506L), M. yongonense (E509H) and M. gastri which had multiple mutations at positions D516V, H526D and, S531F (Table 5). This study established that 23% of NTM harbored mutations associated with resistance to at least one of the antibiotics in the macrolide-based therapy. Regional distribution of NTM with mutations in the target genes had a significant correlation (p=0.012) with the bulk of the isolates originating from the Lake Victoria (n=6, 5%), Coastal (n=6, 5%), and Nairobi (n=6, 5%) regions. The observed regional diversity of NTM harboring drug resistance mutations across Kenya could be attributed to NTM evolution to evade natural antibiotics secreted as secondary metabolites by nearby environmental bacteria in various geographical landscapes (Moore et al., 2019). The 21-35 years age group had the highest number of NTM isolates presenting with drug resistance associated mutations while males comprised the majority (n=21, 76.5%) of cases with NTM showing these mutations. These mutations were associated with a history of previous TB infection as seen in the high number of TB relapse cases recorded in this study (n=8; 28.5%, p=0.001). This is a common observation in sub-Saharan Africa given the high incidence of M. tuberculosis disease and the frequent misdiagnosis of NTM infection with TB seen in this region (    Despite NTM demonstrating high levels of resistance to a broad range of antibiotics, macrolides including clarithromycin, erythromycin, and azithromycin remain the most effective antibiotic with >80% of isolates showing susceptibility (Ananta et al., 2018). However, some NTM including MAC and M. abscessus have been associated with increased resistance to macrolides leading to treatment failure (Saxena et al., 2021). The mechanisms of macrolide resistance have been studied at the molecular level and has consistently demonstrated that 80-100% of phenotypic macrolide resistant clinical isolates contained point mutations at positions A2058 and A2059 in the 23S rRNA gene for NTM (Huh et al., 2019). According to 23S rRNA crystallography investigations, A2058 and A2059 serve as important contact sites for macrolide binding since they are conveniently situated on the surface of the peptide exit tunnel of ribosomes. The architecture of the macrolide binding site is changed by a point mutation of A2058/A2059, which creates a smooth, sunken interaction surface that weakens the interaction of macrolides with their binding site hence leading to drug resistance (Poehlsgaard et al., 2005). Our study demonstrated that majority of macrolide-resistant isolates ( The commonly used aminoglycosides for the treatment of NTM are amikacin, streptomycin, kanamycin, tobramycin, and streptomycin (Krause et al., 2016). In our present study, three M. abscessus subsp abscessus, two M. chelonae, and one M. alsense presented with a A1408G mutation. Genotypic characteristics in rrs that are associated with aminoglycoside resistance usually are in concordance with DST broth microdilution and GenoType NTM-DR assays, implying that mutations within rrs are the molecular basis of aminoglycoside resistance in NTM (Bouzinbi et al., 2020). For instance, a study that selected in-vitro aminoglycoside-resistant M. abscessus and M. chelonae presented an A!G mutation at position 1408 within the rrs upon sequencing. This confirms that a single point mutation at 1408 is adequate to confer high-level aminoglycoside resistance (Nessar et al., 2011). Further, individual mutations at T1406, C1409 and G1491 have also indicated considerable resistance to aminoglycoside in most M. abscessus subspecies (Nessar et al., 2011). Similar to other bacteria, NTM present with low-level aminoglycoside resistance through the production of drugmodifying enzymes including acetyltransferases ( Similar to M. tuberculosis, resistance to rifampicin in NTM is associated with mutations in the 81 bp RRDR corresponding to codons 503 to 533 of the rpoB gene (Saxena et al., 2021;Zhou et al., 2020). Our study identified 11.6% (10/68) NTM with mutations occurring at codon 531 in M. avium, codon 506 in M. intracellulare, 509 in M. yongonense. M. gastri had amino acid substitutions at positions 516, 526, 531 of the rpoB gene. Our findings did not establish mutations outside of the RRDR. However, low-level rifampicin resistance has previously been demonstrated in M. intracellulare with mutations occurring outside the RRDR at position N494S (Park et al., 2020). Broth microdilution analysis which is the gold standard for rifampicin drug resistance testing presents a high minimum inhibition concentration (MIC) for isolates with mutations in the RRDR, hence confirming the role of these mutations in conferring highlevel resistance to rifampicin (Huh et al., 2019). We further demonstrated that M. gastri harbored more than one mutation within the RRDR which is a unique attribute observed for M. kansasii complex species to which M.gastri belongs (Wu et al., 2018). A similar study obtained rifampicin-resistant M. kansasii from clinical isolates and in vitro generated mutant, and demonstrated mutations in codons 513, 526, and 531 of rpoB which is a common pattern in some SGM and M. tuberculosis (Klein et al., 2001).
We found considerable levels of mutations in the drug target genes for macrolides, aminoglycosides and rifampicin in Kenyan NTM. To guide therapy, both species-level identification and drug resistance testing of NTM should be performed before starting treatment for NTM infection.

Conclusion
We demonstrated significant mutations associated with drug resistance for macrolides, aminoglycosides and rifampicin in NTM isolated from symptomatic TB-negative patients in Kenya.
M. abscessus and MAC were the dominant NTM with rrl mutations, and most mutations occurred at position 2058. Majority of RGM had mutation at position 1408 of the rrs gene, while rpoB mutations presented within the RRDR.

Limitations of the study
This study investigated the acquired mechanisms of drug resistance for NTM. Other intrinsic factors apart from drug target gene mutation could influence the sensitivity of NTM to antibiotics.
Despite this limitation, the study documents drug resistance-associated mutation patterns of Kenyan NTM for the first time, and advocates for drug resistance testing before commencement of treatment for NTM infection. One. 2020; 15(9 September 2020): 1-5.

Open Peer Review
Leena Al-Hassan

Version 2
Thank you for revising the manuscript and incorporating the comments into the text. However I would highly suggest that you revise the sections where you correlate the genotype with phenotype, since you have not done the AST (MIC) in this study. If you have not done the AST testing, you cannot state that they are resistant. Your study is primarily genotype based, and you have detected mutations in target resistance determinants, and I therefore suggest you change the wording of the results and discussion to reflect that. These mutations may be silent mutations, and if you do not have phenotype data to support it, you cannot state the fact that they are resistant. You can only say that they harbour the mutations which in previous reports have led to resistance.
These statements "Twenty-eight (23%) of the NTM were resistant to at least one of the antibiotics used in the macrolide-based treatment " and "Ten (14.7%) of the 68 SGM were resistant to rifampicin, with 40 percent having mutations at codon 531 in the rpoB gene" cannot be proven without AST. You should re-word it to say that 23% harboured mutations associated with resistance to macrolide antibiotics.
Same for the conclusion: you have not demonstrated a significant level of drug resistance for macrolides, aminoglycosides and rifampicin in NTM -you have detected mutations in gene targets that have previously been associated with drug resistance.
I hope my point is clear. I am not doubting your results, and I think it's interesting that you have detected these mutations, however it's not scientifically sound to assume resistance based on genotype only, and you must phrase your results and conclusions according to the methods used and data generated. The way it is written now indicates that you have the AST data for the isolates and you are sure that they are rifampicin or aminoglycoside resistant. I would strongly recommend you perform the AST -even for a subset of isolates -to confirm that the observed genotype correlates with the phenotype. The manuscript can be accepted now.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes © 2022 Sengupta U. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The manuscript has been written on the findings of antimycobacterial resistance of NTM isolated from 122 sputum M. tuberculosis-negative patients. The authors have investigated their resistance patterns on the isolated strains using gene sequencing of the drug-resistant genes and noted the mutations in the respective codon positions.

Utpal Sengupta
Materials and methods have been well described. Data were properly analyzed and presented adequately in the tables. However, there were a lot of discrepancies in Table 2 in the first column where the total sample number was shown to be 135 instead of 122 and therefore the percentage expressions were wrongly mentioned in the table and in the text. The percentages have to be calculated correctly and Table 2 has to be modified and described accordingly in the text.
The discussion has been adequate, citing adequate references. However, the discussion with respect to the results of Table 2 has to be modified and discussed for a correct understanding of the reader.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Infectious diseases with special reference to tuberculosis and leprosy, Cell Biology, Microbiology, Immunology and Biotechnology.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.